Chronic Stress Decreases Cerebrovascular Responses During Rat Hindlimb Electrical Stimulation

Abstract

Repeated stress is one of the major risk factors for cerebrovascular disease, including stroke, and vascular dementia. However, the functional alterations in the cerebral hemodynamic response induced by chronic stress have not been clarified. Here, we investigated the in vivo cerebral hemodynamic changes and accompanying cellular and molecular changes in chronically stressed rats. After 3 weeks of restraint stress, the elicitation of stress was verified by behavioral despair in the forced swimming test and by physical indicators of stress. The evoked changes in the cerebral blood volume and pial artery responses following hindpaw electrical stimulation were measured using optical intrinsic signal imaging. We observed that, compared to the control group, animals under chronic restraint stress exhibited a decreased hemodynamic response, with a smaller pial arterial dilation in the somatosensory cortex during hindpaw electrical stimulation. The effect of chronic restraint stress on vasomodulator enzymes, including neuronal nitric oxide synthase (nNOS) and heme oxygenase-2 (HO-2), was assessed in the somatosensory cortex. Chronic restraint stress downregulated nNOS and HO-2 compared to the control group. In addition, we examined the subtypes of cells that can explain the environmental changes due to the decreased vasomodulators. The expression of parvalbumin in GABAergic interneurons and glutamate receptor-1 in neurons were decreased, whereas the microglial activation was increased. Our results suggest that the chronic stress-induced alterations in cerebral vascular function and the modulations of the cellular expression in the neuro-vasomodulatory system may be crucial contributing factors in the development of various vascular-induced conditions in the brain.

Keywords: chronic stress; neurovascular coupling; optical intrinsic signals; restraint stress; sensory stimulation; somatosensory cortex.

Figure 1

Schematic of the experimental design and chronic stress model verification. (A) Experimental protocol. (B) Acquisition of optical intrinsic signal imaging and field of view of the rat sensory cortex [red: hindlimb area (HL), green: forelimb area (FL), blue: shoulder area, purple: barrel cortex (BC)]. (C) Comparisons of the weight gain in the control group and the stress group [Control (n) = 10 and Stress (n) = 10]. (D) Analysis of corticosterone concentrations in the plasma [Control (n) = 10 and Stress (n) = 10]. (E) Behavioral analysis of the forced swimming test [FST, Control (n) = 10 and Stress (n) = 10]. The Mann–Whitney test and independent t-test were performed to analyze the significant differences (^*p < 0.05; ^**p < 0.01; ^***p < 0.001).

Figure 2

Hemodynamic responses in the somatosensory cortex upon 1 mA hindpaw electrical stimulation. Representative images of the hemodynamic responses from a non-stressed rat (A) and a 3-week chronically stressed rat (B). The activation maps show the changes in the cerebral blood flow every other second following hindpaw electrical stimulation using color mapping. (C) The time series analysis of maximum ΔCBV in the control and stressed groups. (D) The time series analysis of the spatial extent of changes surpassing 1% ΔCBV. The red lines on the x-axis in (C,D) indicate the hindpaw stimulation period. The yellow-shaded areas on the plots of (C,D) represent the frames that showed significant changes between the control group and the stress group [Control (n) = 10 and Stress (n) = 10, Mann–Whitney test, p < 0.05].

Figure 3

The process of vessel segmentation used to extract the responsive pial artery to measure the hemodynamic response upon peripheral somatosensory stimulation in the control (A) and 3-week stress (B) groups. The responses of the vessels were displayed as the changes in the number of pixels over the threshold, a standard for distinguishing the vessels from the tissues. The vessel segmentation was performed by combining a Weka segmentation-training method (image J) and standard deviation mapping: (Aa,Ba) raw image, (Ab,Bb) classified images of the vessel and tissue using Weka segmentation method in the raw image (the red region represents the vessel and the green region represents the tissue), (Ac,Bc) vessel probability map, (Ad,Bd) a color-coded standard deviation map (SD map), (Ae,Be) the mask image used to define the “responsive area” based on the SD map, (Af,Bf) the responsive pial artery defined by the mask image overlapping the vessel probability map and the dynamics of the responsive pial arterial diameter, and (Ag,Bg) the non-responding vessels upon electrical stimulation and dynamic changes of the vessel diameters. The red lines on the x-axis in (Af,g,Bf,g) indicate the hindpaw stimulation period. All scale bars under the representative segmentation images indicate 1mm. (C) Comparisons of the dynamics between the pial artery of the control and stress groups in the maximum vessel dilation change (Ca), the time to maximum peak (Cb), the onset time (Cc), and the rising slope (Cd) [Control (n) = 6 and Stress (n) = 5, Independent t-test, ^*p < 0.05].

Figure 4

Protein expression related to vasomodulation in the somatosensory cortex of control and 3-week stress groups. (A,B) Changes in expression of NO- and CO-producing enzymes. The western blot band intensity was normalized to the control by densitometry [nNOS, neuronal nitric oxide synthase; iNOS, inflammatory nitric oxide synthase; eNOS, endothelial nitric oxide synthase; HO-1, Heme oxygenase-1; HO-2, Heme oxygenase-2, Control (n) = 3 and Stress (n) = 3, Independent t-test, ^*p < 0.05; ^**p < 0.01].

Figure 5

Changes in the expression of PV in GABAergic interneurons, GluR-1 in neurons, and microglial activation in the somatosensory cortex of control and 3-week restraint-stressed rats. (A) Representative images of PV-positive cells (green), NPY-positive cells (red), (B) GluR-1-positive cells (green), and (C) Iba1-positive microglial cells (red). All nuclei are stained using DAPI. The images in the white dashed box are magnified in the lower image. The number of PV- labeled cells (D), NPY- labeled cells (E), GluR1- labeled cells (F), Iba1- labeled cells (G), and DAPI- labeled nuclei (H) in both animal groups were counted (PV, parvalbumin; NPY, neuropeptide Y; DAPI, 4′,6-diamidino-2-phenylindole; GluR1, glutamate receptor 1; Iba1, ionized calcium binding adaptor molecule 1, Independent t-test, ^*p < 0.05; ^**p < 0.01; ^***p < 0.001).

Figure 6

Proposed cellular and molecular signaling pathway alterations associated with chronic stress. Theoretical cascade of the effects of chronic stress, including the neurovasomodulatory alterations suggested by these experimental results from the restraint-stressed rats (GR, glucocorticoid receptor).